Endophytic Fungi Isolated from Baccharis linearis and Echinopsis chiloensis with Antifungal Activity against Botrytis cinerea

Botrytis cinerea is one of the most important phytopathogens in agriculture worldwide, infecting economically important crops. The main control of this fungus is by synthetic fungicides, causing the selection of resistant isolates. Compounds produced by endophytic fungi have been shown to have antifungal activity against this pathogen and can be used as an alternative to synthetic fungicides. The aim of this work was to isolate endophytic fungi from Chilean foothills in the Metropolitan Region. Ten fungi were isolated from Echinopsis chiloensis and Baccharis linearis, however, only two isolates inhibited the mycelial growth of B. cinerea by antibiosis and were identified as Epicoccum sp. and Pleosporales sp. Extracts at 200 mg L−1 from Epicoccum sp. and Pleosporales sp. showed antifungal activity against B. cinerea of 54.6 and 44.6% respectively. Active compounds in the Epicoccum sp. extracts were mainly alkaloids and phenolic compounds; meanwhile, in the Pleosporales sp. extracts, terpenes and/or saponins were responsible for the antifungal activity.


Introduction
Botrytis cinerea is a phytopathogenic fungus that produces the disease called gray mold within a wide range of plant species [1] and it is one of the most important plant pathogen fungi globally, causing significant economic losses during storage and transportation [1]. The main control method of this disease is by synthetic compounds [1]. However, this control method affects the environment [2] and the selection of resistant isolates of the fungus [3]. In Chile, B. cinerea resistant isolates have also been described in 'Thompson Seedless' table grapes. Several fungicides were tested and only around 20% of the isolates were sensitive to all these fungicides [4]. Hence, the development of new antifungals to control B. cinerea requires urgent attention.
In recent years, attention has focused on the development of new strategies based on chemical compounds obtained from microorganisms for the control of B. cinerea. The main advantages of these compounds are that they last a short time in the environment and have a varied mechanism of action that prevents the development of resistant strains of the phytopathogen [5].
In the last decade, plant endophytic fungi have increased due to their potential as producers of active secondary metabolites [6]. Most plants are colonized by endophytic fungi; it has been reported that plants are the host of one or several strains of endophytes [7]. These are microorganisms that can live inside tissue of living plants without showing any disease symptoms [8]. Endophytes can produce different secondary metabolites with biological activities such as antibacterial, fungicidal, and algicidal properties [9][10][11]. Different

Isolation of Endophytes
Tissue surface sterilization was carried out as described by Silva-Hughes et al. [26] with modifications. Tissues were immersed in 70% ethanol and 0.2% Tween 20 for three minutes, then washed with sterile distilled water and treated with 2.5% sodium hypochlorite for three minutes. Lastly, tissues were washed with sterile distilled water once more. Sterilized tissues were then cut into small fragments (0.5 cm), placed in Petri dishes containing potato dextrose agar (PDA), and then supplemented with kanamycin sulfate (0.05 mg L −1 ) and chloramphenicol (0.034 mg L −1 ). Plates were incubated for fourteen days at 22 • C. Fungi obtained were transferred to new PDA plates and incubated at 22 • C. This procedure was repeated several times until obtaining a pure culture. The isolated endophytes from E. chiloensis and B. linearis were designated Ech1 to Ech6 and Bl1 to Bl4, respectively.

Antifungal Assays against B. cinerea
The strain G29 of B. cinerea was used in this study. This strain was isolated from infected grapes (Vitis vinifera) [27].

Confrontation Assays
The evaluation of the antifungal activity of the endophytes against B. cinerea was carried out using dual confrontation assays as described by Chen et al. [28], with modifications. Small discs of PDA culture medium with mycelium of the isolated endophytes and B. cinerea were inoculated in opposites sides of PDA plates (6 cm apart) supplemented with antibiotics. B. cinerea, inoculated in one side of the PDA Petri dishes, was used as a control. Inhibition percentage was calculated using the radial mycelial growth of B. cinerea, confronting the endophyte (Ri) and the radial mycelial growth of B. cinerea in the control (Rc) according to the formula (Rc − Ri)/Rc × 100. These experiments were done in triplicate.

Identification of Endophytes
For the molecular identification of the endophytic fungi, genomic DNA was purified using the CTAB method [29]. Around 200 mg of endophyte tissue obtained from axenic cultures was placed in a Falcon tube with 800 µL of CTAB buffer (3% CTAB, 1.4 M NaCl, 20 mM EDTA and 100 mM Tris-HCl pH 8.0). The tissue was vortexed using 4 mm diameter glass spheres for 3 min. Disrupted tissue was incubated at 60 • C for 30 min. Then, it was centrifuged at 8400× g for 10 min at room temperature, and the supernatant was separated from the cellular debris. The recovered supernatant was treated with 2 ng µL −1 RNAse at 37 • C for 30 min. Next, 800 µL of chloroform:isoamyl alcohol (24:1) was added, and the aqueous phase was recovered after centrifugation for 14,196× g for 10 min at room temperature. Later, 800 µL of cold isopropanol was added and incubated at 20 • C for 2 h. Subsequently, the solution was centrifuged for 14,196× g for 10 min at 4 • C. After, 500 µL absolute ethanol was added to the pellet and dried using a paper towel. Finally, DNA was resuspended in DNAse free water.
PCR reaction was performed in 50 µL volumes, containing 2 µL of genomic DNA, 1 µL of forward primer (10 µM), 1 µL of reverse primer (10 µM), 21 µL of nuclease-free water, and 25 µL of GoTaq ® Green Master Mix 2× (Promega, Madison WI, USA). PCR reaction mixtures for amplify ITS region were subjected to an initial denaturation at 94 • C for 3 min, and 38 cycles using the following temperatures: 94 • C for 40 s, 55 • C for 45 s, and 72 • C for 40 s; finally, an elongation was carried out at 72 • C for 5 min. The amplification program for β-tubulin PCR began with an initial denaturation at 94 • C for 5 min and 35 cycles using the following temperatures: 94 • C for 45 s, 55 • C for 45 s, and 72 • C for 2 min; finally, an elongation was carried out at 72 • C for 10 min. PCR reactions were performed in an Eppendorf ® (Hamburg, Germany) MasterCycler Personal. Finally, the PCR products were sequenced by the Genomic and Bioinformatic Center of the Universidad Católica de Chile using automatic sequencing ABI PRISM 3100.
Electropherograms were manually edited using Geneious Prime ® (Biomatters Ltd., Auckland, New Zealand) 2021.01 version software. A consensus sequence was created using both ITS and β-tubulin sequences, BLASTn tool was used to look for similarities with other fungi. Finally, using Geneious Prime software, consensus sequences tree construction was carried out using sequences obtained using ClustalW algorithm. Phylogenetic trees were constructed using the Tamura-Nei genetic distance model using the neighbor-joining method. Resampling for each dendrogram was 1000 using Geneious Prime software.

Extraction of Secondary Metabolites from the Most Active Endophytic Fungi
For the extraction of secondary metabolites, fungi were inoculated in PDA plates on a cellophane layer and incubated for seven days. After this, cellophane containing the fungi was discarded. Remaining culture medium was extracted using ethyl acetate. The organic phase was evaporated using a rotary evaporator at 40 • C. PDA without endophyte was used as negative control.

Antifungal Activity against B. Cinerea of the Extracts
The antifungal activity of the extracts was evaluated in vitro. Extracts dissolved in acetone were added to PDA plates at 50, 100, and 200 mg L −1 . Acetone was added to PDA plates as a negative control. The final acetone concentration was identical in the control and treatment assay. After acetone evaporation in a laminar flow cabinet, the culture media were inoculated with 0.5 cm agar disks from an active growing culture of B. cinerea. Cultures were incubated in the dark at 22 • C for three days. Mycelium diameter was measured daily in two perpendicular directions. Inhibition percentages were calculated after 72 h of incubation. These experiments were done in triplicate.
A bioautography was carried out using thin-layer chromatography (TLC) to identify what fraction of the extract containing the diffusible compounds had antifungal activity [33,34]. Extract dissolved in methanol was placed in a TLC plate (silica gel 60 F254, Merck, Santiago, Chile) and separated with methanol:chloroform (9:1) as an eluent system. The separation of the compound in the TLC was visualized using UV light at 254 nm. Mobile phase methanol:chloroform (9:1) was placed in TLC plate as solvent control. Bioautography using the TLC with the separated compounds was carried out as described by Vidal et al. [11].
A preliminary characterization of the antifungal compounds in the extracts from the isolates Ech4 and Bl1 was performed using different stain solutions. Extracts were separated in TLC plates using chloroform:methanol (9:1) as an eluent system and stained with the following solutions: sulfuric acid (25% v/v) for organic compounds, Dragendorff's reagent spray solution (Merck) for alkaloids, solution of iron (III) chloride (2% w/v iron chloride, methanol 50% v/v and 50% v/v water) for phenolics compounds, a solution of vanillin-sulfuric acid (3.5% w/v vanillin in methanol and 0.625% v/v of sulfuric acid) for terpenoids [35] and saponins [36].

Statistical Analysis
For all the non-parametrical statistical analyses, GraphPad Prism 6.01 was used. Statistical significance between treated groups and the control group by multiple t-tests and Holm-Sidak method are indicated with asterisks (p < 0.05).

Isolation of Endophytes
Endophytic fungi were isolated from root of the endemic plant Echinopsis chiloensis and the native plant Baccharis linearis ( Figure 1).
Six endophytic fungi were isolated from apparently healthy E. chiloensis and four fungi from B. linearis (Table 1).
Evaluation of the antifungal activity against B. cinerea of the isolated fungi was carried out by using the dual confrontation assay. Among these, only two isolates, Bl1 and Ech4, showed a significative inhibition of the mycelial growth of B. cinerea Six endophytic fungi were isolated from apparently healthy E. chiloensis a fungi from B. linearis (Table 1).

Plant.
Fungal Isolate Evaluation of the antifungal activity against B. cinerea of the isolated fungi w ried out by using the dual confrontation assay. Among these, only two isolates, Ech4, showed a significative inhibition of the mycelial growth of B. cinerea (p < 0. calculated inhibition values were 26.4 and 44.0% for Bl1 and Ech4, respectively (F

Plant.
Fungal Isolate Evaluation of the antifungal activity against B. cinerea of the isolated fu ried out by using the dual confrontation assay. Among these, only two isol Ech4, showed a significative inhibition of the mycelial growth of B. cinerea (p calculated inhibition values were 26.4 and 44.0% for Bl1 and Ech4, respective  Each bar represents the average inhibition percentage of three experiments ± SD. Statistical significance when comparing treated groups and control group by multiple t-tests and Holm-Sidak method are indicated with asterisks (p < 0.05). Table 1. Endophytes isolated from the recollected plants.

Plant.
Fungal Isolate The formation of an inhibition halo between the mycelia of both isolates, Ech4 and Bl1, and B. cinerea ( Figure 3) suggests that the antifungal effect was produced by diffusible compounds (antibiosis) secreted by Ech4 and Bl4.
The formation of an inhibition halo between the mycelia of both isolates, Ech4 and Bl1, and B. cinerea ( Figure 3) suggests that the antifungal effect was produced by diffusible compounds (antibiosis) secreted by Ech4 and Bl4.

Antifungal Activity against B. cinerea of the Extracts
Extracts of the secondary metabolites produced from Ech4 and Bl1 were obtained, and the antifungal activity of these extracts was evaluated against B. cinerea ( Figure 4). The extract obtained from the endophytic fungus Bl1 showed a higher antifungal activity against B. cinerea than the extract obtained from the endophytic fungus Ech4 at the tested concentrations. The extracts from Bl1 and Ech4 showed a higher antifungal activity at 200 mg L −1 , being 54.6 and 44.6%, respectively. Additionally, to identify what fraction of the extracts was responsible for the antifungal activity, a bioautography assay was carried out using 2 mg of both extracts ( Figure 5). Results showed that not all the compounds in the extracts had antifungal activity. Extract of the isolate Bl1 showed an inhibition halo for compounds with lower Rf values; meanwhile, the extract obtained from Ech4 also showed an inhibition halo in the zone of the compounds with higher Rf values ( Figure 5).

Antifungal Activity against B. cinerea of the Extracts
Extracts of the secondary metabolites produced from Ech4 and Bl1 were obtained, and the antifungal activity of these extracts was evaluated against B. cinerea ( Figure 4). The extract obtained from the endophytic fungus Bl1 showed a higher antifungal activity against B. cinerea than the extract obtained from the endophytic fungus Ech4 at the tested concentrations. The extracts from Bl1 and Ech4 showed a higher antifungal activity at 200 mg L −1 , being 54.6 and 44.6%, respectively.

Antifungal Activity against B. cinerea of the Extracts
Extracts of the secondary metabolites produced from Ech4 and Bl1 we and the antifungal activity of these extracts was evaluated against B. cinere The extract obtained from the endophytic fungus Bl1 showed a higher antifu against B. cinerea than the extract obtained from the endophytic fungus Ech4 concentrations. The extracts from Bl1 and Ech4 showed a higher antifungal ac mg L −1 , being 54.6 and 44.6%, respectively. Additionally, to identify what fraction of the extracts was responsible for gal activity, a bioautography assay was carried out using 2 mg of both extrac Results showed that not all the compounds in the extracts had antifungal acti of the isolate Bl1 showed an inhibition halo for compounds with lower Rf va while, the extract obtained from Ech4 also showed an inhibition halo in the compounds with higher Rf values ( Figure 5). Additionally, to identify what fraction of the extracts was responsible for the antifungal activity, a bioautography assay was carried out using 2 mg of both extracts ( Figure 5). Results showed that not all the compounds in the extracts had antifungal activity. Extract of the isolate Bl1 showed an inhibition halo for compounds with lower Rf values; meanwhile, the extract obtained from Ech4 also showed an inhibition halo in the zone of the compounds with higher Rf values ( Figure 5). For the preliminary characterization of the active compounds, different stain solutions were used. The bioactive fraction of the Bl1 extract was positive for Dragendorff's reagent and iron chloride (III) solution, therefore this fraction would contain compounds of the alkaloid family, with the presence of phenolic groups, while the bioactive fraction of extract from Ech4 was positive for the vanillin/sulfuric acid solution, indicating that it could contain terpenes and saponins (Results not shown).

Identification of Endophytes with Antifungal Activity
For the morphological identification, axenic cultures of both endophytes (Ech4 and Bl1) were observed with or without a lactophenol cotton blue stain under a light microscope. The isolate Bl1 developed orange-pigmented, septate vegetative hyphae with branched growth. A single globular orange conidia was developed in each sporodochium. (Figure 6). On the other hand, the isolate Ech4 showed in PDA medium a branched hyaline septate hypha, yellow-pigmented conidiomata pycnidial globose, covered with some hyphal outgrowths and ovoid shaped small conidia smooth-and thin-walled, hyaline, aseptate. (Figure 7).  For the preliminary characterization of the active compounds, different stain solutions were used. The bioactive fraction of the Bl1 extract was positive for Dragendorff's reagent and iron chloride (III) solution, therefore this fraction would contain compounds of the alkaloid family, with the presence of phenolic groups, while the bioactive fraction of extract from Ech4 was positive for the vanillin/sulfuric acid solution, indicating that it could contain terpenes and saponins (Results not shown).

Identification of Endophytes with Antifungal Activity
For the morphological identification, axenic cultures of both endophytes (Ech4 and Bl1) were observed with or without a lactophenol cotton blue stain under a light microscope. The isolate Bl1 developed orange-pigmented, septate vegetative hyphae with branched growth. A single globular orange conidia was developed in each sporodochium. (Figure 6). On the other hand, the isolate Ech4 showed in PDA medium a branched hyaline septate hypha, yellow-pigmented conidiomata pycnidial globose, covered with some hyphal outgrowths and ovoid shaped small conidia smooth-and thin-walled, hyaline, aseptate. (Figure 7).  For the preliminary characterization of the active compounds, different stain solutions were used. The bioactive fraction of the Bl1 extract was positive for Dragendorff's reagent and iron chloride (III) solution, therefore this fraction would contain compounds of the alkaloid family, with the presence of phenolic groups, while the bioactive fraction of extract from Ech4 was positive for the vanillin/sulfuric acid solution, indicating that it could contain terpenes and saponins (Results not shown).

Identification of Endophytes with Antifungal Activity
For the morphological identification, axenic cultures of both endophytes (Ech4 and Bl1) were observed with or without a lactophenol cotton blue stain under a light microscope. The isolate Bl1 developed orange-pigmented, septate vegetative hyphae with branched growth. A single globular orange conidia was developed in each sporodochium. (Figure 6). On the other hand, the isolate Ech4 showed in PDA medium a branched hyaline septate hypha, yellow-pigmented conidiomata pycnidial globose, covered with some hyphal outgrowths and ovoid shaped small conidia smooth-and thin-walled, hyaline, aseptate. (Figure 7).   For the molecular identification, the ITS region and β-tubulin marker were amplified for both Ech4 and Bl1 isolates. PCR products for the ITS sequence are shown as bands arounds 500 bp and 600 bp in the agarose gel and β-tubulin sequence shown as bands arounds 300 bp. Most similar ITS and β-tubulin sequences to the isolates were searched by an alignment using the NCBI BLASTn tool. The phylogenetic tree, based on ITS and βtubulin sequences, showed isolate Bl1 and Epicoccum spp. are clustered in the same clade (Figures 8 and 9). On the other hand, the phylogenetic tree based on ITS and β-tubulin showed that the isolate Ech4 is clustered in the same clade with more than one family, such as Cucurbitariaceae, Leptosphaeriaceae, and Pleosporaceae (Figures 10 and 11).  For the molecular identification, the ITS region and β-tubulin marker were amplified for both Ech4 and Bl1 isolates. PCR products for the ITS sequence are shown as bands arounds 500 bp and 600 bp in the agarose gel and β-tubulin sequence shown as bands arounds 300 bp. Most similar ITS and β-tubulin sequences to the isolates were searched by an alignment using the NCBI BLASTn tool. The phylogenetic tree, based on ITS and β-tubulin sequences, showed isolate Bl1 and Epicoccum spp. are clustered in the same clade (Figures 8 and 9). On the other hand, the phylogenetic tree based on ITS and β-tubulin showed that the isolate Ech4 is clustered in the same clade with more than one family, such as Cucurbitariaceae, Leptosphaeriaceae, and Pleosporaceae (Figures 10 and 11). For the molecular identification, the ITS region and β-tubulin marker were amplified for both Ech4 and Bl1 isolates. PCR products for the ITS sequence are shown as bands arounds 500 bp and 600 bp in the agarose gel and β-tubulin sequence shown as bands arounds 300 bp. Most similar ITS and β-tubulin sequences to the isolates were searched by an alignment using the NCBI BLASTn tool. The phylogenetic tree, based on ITS and βtubulin sequences, showed isolate Bl1 and Epicoccum spp. are clustered in the same clade (Figures 8 and 9). On the other hand, the phylogenetic tree based on ITS and β-tubulin showed that the isolate Ech4 is clustered in the same clade with more than one family, such as Cucurbitariaceae, Leptosphaeriaceae, and Pleosporaceae (Figures 10 and 11).

Discussion
In this work, ten endophytic fungi were found in roots from plants growing in Chilean Central Precordillera. Six endophytes were found from the endemic plant E. chiloensis. Previously, an endophytic fungus belonging to the genus Alternaria was isolated from the

Discussion
In this work, ten endophytic fungi were found in roots from plants growing in Chilean Central Precordillera. Six endophytes were found from the endemic plant E. chiloensis. Previously, an endophytic fungus belonging to the genus Alternaria was isolated from the mesenchymal tissue of this plant that inhabited in the same location [11]. This difference could be explained because the diversity of fungi occurring in shoots and roots may have important variations [37]. In addition, mycelial growth within plant tissues is heterogeneous [38], and the presence of endophytes and the synthesis of their metabolites may have marked seasonal variations [39,40]. Also, various isolates from the same fungal specie, obtained from separate plant sources, can produce different secondary metabolites [41].
On the other hand, in the endemic plant B. linearis, four endophytes were found, and this is the first report of endophytic fungi isolated from this plant.
Consequently, ten endophytes were isolated in this work, a similar number as other studies. For example, fourteen endophytic fungi were isolated from five endemic plants in India using a similar isolation method [42], and around five endophytes per plant from different gymnosperm plants were found in Chile [17]. On the other hand, also a higher number of endophytes have been reported in other studies, for example, a 108 endophytes were obtained from the endemic plant Opuntia humifusa in the United States [26], and 319 fungal species were isolated from the roots of 24 plant species from Spain [43]. Nevertheless, the relatively low number of endophytes found in this study could be explained by a rigorous disinfection of the plant surface or by different growing conditions, for instance, Silva-Hughes et al. incubated plant fragments for 60 days [26], an incubation period around four times longer than in this work. Out of ten endophytic fungi, two fungi designated as Bl1 and Ech4 showed antifungal activity against B. cinerea in dual confrontation assays; this inhibition was similar to an endophytic Alternaria sp. against the same isolate of B. cinerea using the same experimental conditions [11].Other authors previously reported endophytic fungi with antifungal activity against B. cinerea in Chile, such as endophytes found in the endemic tree Embotrium coccineum [18], endophytic fungi isolated from Artemisia absinthium [44], Penicillium janczewskii and Microsphaeropsis olivacea, endophytes isolated from Chilean native gymnosperms [17], and Alternaria sp. and Aureobasidium sp. fungi isolated from Chilean endemic and native plants [11]. This suggests that the fungi found could have potential as a biocontrol agent of this phytopathogen.
In addition, it was shown that both Bl1 and Ech4 isolates inhibited the B. cinerea mycelial growth by antibiosis, similar to other endophytic fungi; for example, fungi isolated from Theobroma cacao showed antibiosis against pathogens such as Moniliophthora roreri and Phytophthora palmivora [45], and endophytic fungi isolated from Aloe vera also inhibited by antibiosis the mycelial growth of Fusarium oxysporum [46]. The inhibition halo found in both isolates indicates that the antifungal activity of the obtained endophytic fungi is due to the secretion of diffusible secondary metabolites to the culture medium.
The identification of the Bl1 and Ech4 isolates by phylogenetic analysis based on the molecular markers ITS-rDNA and β-tubulin suggests that both endophytes are grouped with species of the order Pleosporales. The order of the Pleosporales is one of the largest of Dothideomycetes [47]. In this order, families such as Didymellaceae, Didymosphaeriaceae, Pleosporaceae, Hypsostromataceae, Cucurbitariacea, and others have been identified, and, within this classification, epiphytic, endophytic, and phytopathogenic fungal species have been found [48]. In detail, the sequences of the isolate Bl1 are more closely grouped with the genus Epicoccum, while the Ech4 isolate is closely grouped with more than one family within the suborder Pleosporineae [48].
On the other hand, in the case of isolate Bl1, this grouping is confirmed with the morphological analysis of the axenic culture, where the presence of sporodochia with orange-pigmented conidiomata are characteristic to species belonging to the genus Epicoccum, such as E. italicum, E. layuense, and E. poae, and the difference between these species is the conidium size [49,50]. Epicoccum species have been extensively reviewed as ubiquitous phytopathogens that can be used as biological control agents [51]. Epicoccum spp. have been found in different plant parts [50] and may be endophytes [52] and even saprophytes or pathogens [53,54].
Axenic culture of the isolate Ech4 showed small ovoid-shaped conidia and pigmented pilose pycnidia, which is described for different species of the Pleosporales order, such as the Phoma genus [49,55,56]. However, Phoma is considered a group of paraphyletic species that are continuously renamed and reassigned to different families of the Pleosporinae suborder, in addition to some genera whose family are still considered incertae sedis [47,48,57]. Additionally, Hou et al. suggested that the use of conventional molecular markers such as ITS and β-Tubulin present limitations that can be improved with the use of the rpb2 molecular marker to arrive at more reliable phylogenies [58]. Thus, in this study it is only established that isolate Ech4 corresponds to the Pleosporales order.
Many studies have been described that fungi of the order Pleosporales and the genus Epicoccum have been isolated as endophytes and their antifungal activity against different phytopathogenic fungi has been demonstrated [59][60][61][62]. This is the first time that Epicoccum sp. have been isolated from E. chiloensis and that an endophytic fungus of the order Pleosporales have been isolated from B. linearis.
On the other hand, in this work, the results suggest that both extracts obtained from the fungal isolates inhibit the mycelial growth of B. cinerea. Compounds with antimicrobial activity have been described for Epicoccum sp. and for many other Pleosporales species, such as Coniothyrium sp. and Phoma sp. [13,63,64]. Bioautography assays results suggest that orange active fraction of the extract from the isolate Bl1 (Epicoccum sp.), belongs to a group of pigmented compounds such as polyketides, carotenoids, and flavonoids already described in the genus Epicoccum [65]. Preliminary characterization of active fractions of the extract of the Epicoccum sp. isolate presented positive reaction with Dragendorff's stain and iron (III) chloride solution, suggesting the presence of alkaloids and phenolics compounds in the extract. Alkaloids such as epicorazine A and epicorazine B have been described for this fungus and they have shown antimicrobial activity [66]. Also, aromatic polyketides with antifungal properties have been previously found in the Epicoccum spp. [67]. Meanwhile, active fraction of the extract from the isolate Ech4 suggest the presence of antifungal compounds. Some Pleosporales species of endophytic fungi such as Phoma sp. [59] and Alternaria sp. [11] produce metabolites with antifungal activity against B. cinerea [11,68]. Since the extract from Ech4 only showed a positive reaction for the vanillin/sulfuric acid stain, this suggests the presence of terpenes and their derivatives, such as saponins [69,70]. It has been reported that similar compounds have been described for fungal species of the order Pleosporales, such as Leptosphaeria sp. producing triterpenoid saponin to improve damage in their host, Dipsacus asperoides [71], and Phoma sp. producing terpenes, such as aphidicolin, a specific inhibitor of DNA polymerase, as well as antifungal compounds such as furan and dihydrofuran derivatives [64,72].
In conclusion, two endophytic fungi, Epicoccum sp. and Pleosporales sp., with antifungal activity against B. cinerea were isolated from the roots of plants growing in the Chilean Central Precordillera. This is the first report of Epicoccum sp. being isolated from E. chiloensis and of an endophytic fungus of the order Pleosporales being isolated from B. linearis. Also, possibly alkaloids and phenolic compounds were found in the extract from Epicoccum sp., and terpenes and their derivate compounds such as saponins from the fungus Pleosporales sp., could be responsible for the antifungal activity against B. cinerea, further study of these compounds and their effects may be a new alternative for the biocontrol of phytopathogens.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.